Chaperone Proteins
The molecular chaperones of the Heat Shock Proteins class (HSPs), which are classified according to their molecular weight (Hsp27, Hsp70, Hsp90 etc.), are key elements in the balance between the synthesis and degradation of cellular proteins that are responsible for correct protein folding. They play a vital role in the response to cellular stress. The HSPs, and in particular Hsp90, are also involved in the regulation of various important cellular functions, via their association with various client proteins involved in cellular proliferation or in apoptosis (Jolly C. and Morimoto R. I., J. N. Cancer Inst., (2000), 92, 1564-72; Smith D. F. et al., Pharmacological Rev. (1998), 50, 493-513; Smith D. F., Molecular Chaperones in the Cell, 165-178, Oxford University Press 2001).
Hsp90 Chaperone and Hsp90 Inhibitors in the Treatment of Cancers:
The Hsp90 chaperone, which represents 1 to 2% of the protein content of the cell, has recently been shown to be a particularly promising target in anticancer therapy (cf. for a review: Moloney A. and Workman P., Expert Opin. Biol. Ther. (2002), 2(1), 3-24; Chiosis et al., Drug Discovery Today (2004), 9, 881-888). There is interest in particular in the cytoplasmic interactions of Hsp90 with the main client proteins of Hsp90—proteins which are involved in the six mechanisms of tumour progression, as defined by Hanahan D. and Weinberg R. A. (Cell (2002), 100, 57-70), namely:                ability to proliferate in the absence of growth factors: EGFR-R/HER2, Src, Akt, Raf, MEK, Bcr-Abl, Flt-3 etc.        ability to evade apoptosis: mutated form of p53, Akt, surviving etc.        insensitivity to proliferation stop signals: Cdk4, Plk, Wee1 etc.        ability to activate angiogenesis: VEGF-R, FAK, HIF-1, Akt etc.        ability to proliferate without replicative limit: hTert etc.        ability to invade new tissues and to metastasize: c-Met        
Among the other client proteins of Hsp90, steroid hormone receptors, such as the oestrogen receptor or the androgen receptor, are also of considerable interest in connection with anticancer therapies.
It was shown recently that the alpha form of Hsp90 also has an extracellular role via its interaction with the metalloprotease MMP-2, which is itself implicated in tumoral invasion (Eustace B. K. et al., Nature Cell Biology (2004), 6, 507-514).
Hsp90 is composed of two N- and C-terminal domains separated by a highly charged region. Dynamic interaction between these two domains, coordinated by the fixation of nucleotides and of co-chaperones, determines the conformation of the chaperone and its state of activation. Association of the client proteins depends mainly on the nature of the co-chaperones Hsp70/Hsp40, Hop60 etc., and on the nature of the ADP or ATP nucleotide joined to the N-terminal domain of Hsp90. Thus, hydrolysis of ATP to ADP and the ADP/ATP exchange factor control all of the chaperone “machinery”, and it has been shown that it is sufficient to prevent the hydrolysis of ATP to ADP—ATPase activity of Hsp90 —in order to release client proteins in the cytoplasm, which will then be degraded to the proteasome (Neckers L and Neckers K, Expert Opin. Emerging Drugs (2002), 7, 277-288; Neckers L, Current Medicinal Chemistry, (2003), 10, 733-739; Piper P. W., Current Opin. Invest. New Drugs (2001), 2, 1606-1610).
Role of Hsp90 and its Inhibitors in Pathologies Other than Cancer:
Various human pathologies are the consequence of incorrect folding of key proteins, notably leading to neurodegenerative diseases following aggregation of certain proteins such as in Alzheimer's disease and Huntington's disease or diseases associated with prions (Tytell M. and Hooper P. L., Emerging Ther. Targets (2001), 5, 267-287). In these pathologies, approaches aiming to inhibit Hsp90 in order to activate the stress pathways (Hsp70 for example) might be beneficial (Nature Reviews Neuroscience 6: 11, 2005). Some examples are given below:                i) Huntington's disease: This neurodegenerative disease is due to extension of CAG triplets in exon 1 of the gene encoding the protein huntingtin. It has been shown that geldanamycin inhibits the aggregation of this protein owing to overexpression of the Hsp70 and Hsp40 chaperones (Human Molecular Genetic 10: 1307, 2001).        ii) Parkinson's disease: This disease is due to the progressive loss of dopaminergic neurons and is characterized by the aggregation of the protein alpha-synuclein. It has been shown that geldanamycin is able to protect drosophila against the toxicity of alpha-synuclein on the dopaminergic neurons.        iii) Focal cerebral ischaemia: It was shown in a rat animal model that geldanamycin protects the brain against cerebral ischaemia, through the effect of stimulation of transcription of the genes encoding the “heat-shock proteins” by an Hsp90 inhibitor.        iv) Alzheimer's disease and multiple sclerosis: These diseases are partly due to the expression of proinflammatory cytokines and of the inducible form of NOS (nitric-oxide synthase) in the brain, and this deleterious expression is suppressed by the response to stress. In particular, Hsp90 inhibitors are able to store up this response to stress, and it has been shown in vitro that geldanamycin and 17-AAG display anti-inflammatory activity in the brain's glial cells (J. Neuroscience Res. 67: 461, 2002).        v) Amyotrophic lateral sclerosis: This neurodegenerative disease is due to the progressive loss of motor neurons. It has been shown that arimoclomol, a heat-shock protein inducer, slows down the evolution of the disease in an animal model (Nature Medicine 10: 402, 2004). Since an Hsp90 inhibitor is also an inducer of heat-shock proteins (Mol. Cell. Biol. 19: 8033, 1999; Mol. Cell. Biol. 18: 4949, 1998), it is probable that a beneficial effect might also be obtained in this pathology for inhibitors of this type.        
Moreover, an inhibitor of the Hsp90 protein might potentially be useful in various diseases, other than cancer as already mentioned, such as parasitic, viral or fungal infections, or neurodegenerative diseases—by direct action on Hsp90 and particular client proteins. Some examples are presented below:                vi) Malaria: the Hsp90 protein of Plasmodium falciparum displays 59% identity and 69% similarity with the Human Hsp90 protein, and it has been shown that geldanamycin inhibits the growth of the parasite in vitro (Malaria Journal 2: 30, 2003; J. Biol. Chem. 278: 18336, 2003; J. Biol. Chem. 279: 46692, 2004).        vii) Brugian and bancroftian filariodes: these filarial lymphatic parasites possess an Hsp90 protein that can potentially be inhibited by inhibitors of the human protein. In fact, it has been shown for another similar parasite, Brugia pahangi, that the latter is susceptible to inhibition by geldanamycin. The B. pahangi sequences and human sequences are 80% identical and 87% similar. (Int. J. for Parasitology 35: 627, 2005)        viii) Toxoplasmosis: Toxoplasma gondii, the parasite responsible for toxoplasmosis, possesses an Hsp90 chaperone protein, for which induction has been demonstrated in the course of tachyzoite-bradyzoite conversion, corresponding to transition of the chronic infection to active toxoplasmosis. Moreover, geldanamycin blocks this tachyzoite-bradyzoite conversion in vitro (J. Mol. Biol. 350: 723, 2005)        ix) Mycoses that are resistant to treatment: It is possible that the Hsp90 protein potentiates the development of drug resistance, by allowing new mutations to develop. Consequently, an Hsp90 inhibitor, alone or in combination with another antifungal treatment, might prove to be useful in the treatment of some resistant strains (Science 309: 2185, 2005). Moreover, the anti-Hsp90 antibody developed by Neu Tec Pharma displays activity against C. albicans which is fluconazole-sensitive and fluconazole-resistant, C. krusei, C. tropicalis, C. glabrata, C. lusitaniae and C. parapsilosis in vivo (Current Molecular Medicine 5: 403, 2005).        x) Hepatitis B: Hsp90 is one of the host proteins interacting with the reverse transcriptase of the hepatitis B virus during the viral replication cycle. It has been shown that geldanamycin inhibits the replication of viral DNA and the encapsulation of viral RNA (Proc. Natl. Acad. Sci. USA 93: 1060, 1996)        xi) Hepatitis C: The human Hsp90 protein takes part in the cleavage stage between the NS2 and NS3 proteins by the viral protease. Geldanamycin and radicicol are able to inhibit this NS2/3 cleavage in vitro (Proc. Natl. Acad. Sci. USA 98: 13931, 2001)        xii) Herpes virus: Geldanamycin has demonstrated activity in inhibition of replication of the HSV-1 virus in vitro, with a good therapeutic index (Antimicrobial Agents and Chemotherapy 48: 867, 2004). The authors also found geldanamycin to be active against other viruses HSV-2, VSV, Cox B3, HIV-1 and the SARS coronavirus (data not shown).        xiii) Dengue (or breakbone fever): It has been shown that the human Hsp90 protein takes part in the viral entry stage, forming a complex that also contains Hsp70 which serves as a virus receptor. An anti-Hsp90 antibody reduces the infectiousness of the virus in vitro (J. of Virology 79: 4557, 2005)        xiv) Spinal and bulbar muscular atrophy (SBMA): a hereditary neurodegenerative disease characterized by an extension of CAG triplets in the gene of the androgen receptor. It has been shown that 17-AAG, a derivative of geldanamycin, displays activity in vivo on transgenic animals serving as experimental models of this disease (Nature Medicine 11: 1088, 2005).Hsp90 inhibitors:        
The first known inhibitors of Hsp90 are compounds of the amsamycin family, in particular geldanamycin (1) and herbimycin A. X-ray studies have shown that geldanamycin binds to the ATP site of the N-terminal domain of Hsp90, where it inhibits the ATPase activity of the chaperone (Prodromou C. et al., Cell (1997), 90, 65-75).
The NIH and Kosan BioSciences are currently funding the clinical development of 17-AAG (2), an Hsp90 inhibitor derived from geldanamycin (1), which blocks the ATPase activity of Hsp90 by binding to the N-terminal ATP recognition site. Based on the results of phase I clinical trials of 17-AAG (1), phase II trials are now beginning, but research is also being directed towards derivatives that are more soluble such as analogue 3 (17-DMAG from Kosan BioSciences), which bears a dimethylamine chain instead of the methoxy residue, and towards optimized formulations of 17AAG (CNF1010 from Conforma Therapeutics):

The reduced analogue of 17-AAG (WO 2005063714/US 2006019941) has also recently entered phase I clinical studies by the company Infinity Pharmaceuticals. Novel derivatives of geldanamycin have been described recently (WO2006016773/U.S. Pat. No. 6,855,705/US 2005026894/WO2006/050477/US 2006205705).
Radicicol (4) is also an Hsp90 inhibitor of natural origin (Roe S. M. et al., J. Med. Chem. (1999), 42, 260-66). However, although it is by far the best inhibitor of Hsp90 in vitro, its metabolic instability with respect to sulphur-containing nucleophiles makes it difficult to use in vivo. Oxime derivatives that are much more stable such as KF 55823 (5) or KF 25706 have been developed by the company Kyowa Hakko Kogyo (Soga et al., Cancer Research (1999), 59, 2931-2938)

Structures of natural origin related to radicicol have also been described recently, such as zearalenone (6) by the company Conforma Therapeutics (WO 03041643) or compounds (7-9).

Patent application US2006089495 describes mixed compounds comprising a quinone nucleus, such as amsamycin derivatives, and a resorcinol nucleus such as the analogues of radicicol, as Hsp90 inhibitors.
An Hsp90 inhibitor of natural origin, novobiocin (10), binds to a different ATP site located in the C-terminal domain of the protein (Itoh H. et al., Biochem J. (1999), 343, 697-703. Recently, simplified analogues of novobiocin have been identified as more potent Hsp90 inhibitors than novobiocin itself (J. Amer. Chem. Soc. (2005), 127(37), 12778-12779).

Patent application WO 2006/050501 claims analogues of novobiocin as Hsp90 inhibitors.
A depsipeptide, called Pipalamycin or ICI101 has also been described as a non-competitive inhibitor of the ATP site of Hsp90 (J. Pharmacol. Exp. Ther. (2004), 310, 1288-1295).
Sherperdine, nonapeptide KHSSGCAFL, mimics a portion of the K79-K90 sequence (KHSSGCAFLSVK) of survivin and blocks the interaction of proteins of the IAP family with Hsp90 in vitro (WO 2006014744).
Small peptides, comprising a sequence of the Otoferline type (YSLPGYMVKKLLGA), have recently been described as Hsp90 inhibitors (WO 2005072766).
Purines, such as the compounds PU3 (11) (Chiosis et al., Chem. Biol. (2001), 8, 289-299) and PU24FCI (12) (Chiosis et al., Curr. Canc. Drug
Targets (2003), 3, 371-376; WO 2002/036075) have also been described as Hsp90 inhibitors:
A purine derivative CNF2024 (13) was recently introduced into clinical practice by the company Conforma Therapeutics, in collaboration with the Sloan Kettering Memorial Institute for Cancer Research (WO 2006/084030).

Patent application FR2880540 (Aventis) claims another family of purines as Hsp90 inhibitors.
Patent application WO2004/072080 (Cellular Genomics) claims a family of 8-heteroaryl-6-phenyl-imidazo[1,2-a]pyrazines as modulators of Hsp90 activity.
Patent application WO2005/028434 (Conforma Therapeutics) claims aminopurines, aminopyrrolopyrimidines, aminopyrazolopyrimidines and aminotriazolopyrimidines as Hsp90 inhibitors.
Patent application WO2004/050087 (Ribotarget/Vernalis) claims a family of pyrazoles that can be used for treating pathologies associated with inhibition of the heat-shock proteins such as the Hsp90 chaperone.
Patent application WO2004/056782 (Vernalis) claims a novel family of pyrazoles that can be used for treating pathologies associated with inhibition of the heat-shock proteins such as the Hsp90 chaperone.
Patent application WO2004/07051 (Vernalis) claims arylisoxazole derivatives that can be used for treating pathologies associated with inhibition of the heat-shock proteins such as the Hsp90 chaperone.
Patent application WO2004/096212 (Vernalis) claims a third family of pyrazoles that can be used for treating pathologies associated with inhibition of the heat-shock proteins such as the Hsp90 chaperone.
Patent application WO2005/00300 (Vernalis) claims, more generally, 5-membered heterocycles, substituted with aryl radicals, that can be used for treating pathologies associated with inhibition of the heat-shock proteins such as the Hsp90 chaperone.
Patent application JP2005/225787 (Nippon Kayaku) claims another family of pyrazoles as Hsp90 inhibitors.
Application WO2006/018082 (Merck) claims another family of pyrazoles as Hsp90 inhibitors.
Patent application WO2005/00778 (Kyowa Hakko Kogyo) claims a family of benzophenone derivatives as Hsp90 inhibitors, useful for the treatment of tumours.
Patent application WO2005/06322 (Kyowa Hakko Kogyo) claims a family of resorcinol derivatives as Hsp90 inhibitors.
Patent application WO2005/051808 (Kyowa Hakko Kogyo) claims a family of derivatives of resorcinyl-benzoic acids as Hsp90 inhibitors. Patent applications WO2005/021552, WO2005/0034950, WO2006/08503, WO2006/079789 and WO2006/090094 (Vernalis) claim families of pyrimidothiophenes or pyridothiophenes that can be used for treating pathologies associated with inhibition of the heat-shock proteins such as the Hsp90 chaperone.
Application WO2006/010595 (Novartis) claims a family of indazoles as Hsp90 inhibitors.
Application WO2006/010594 (Novartis) claims a family of dihydrobenzimidazolones as Hsp90 inhibitors.
Patent application WO2006/055760 (Synta Pharma) claims a family of diaryl-triazoles as Hsp90 inhibitors.
Patent application WO2006/087077 (Merck) claims a family of (s-triazol-3-yl)phenols as Hsp90 inhibitors.
Patent application FR2882361 (Aventis) claims a family of 3-aryl-1,2-benzisoxazoles as Hsp90 inhibitors.
Patent application WO2006/091963 (Serenex) claims families of tetrahydroindolones and tetrahydroindazolone as Hsp90 inhibitors.
Patent application DE10200509440 (Merck) claims a family of thienopyridines as Hsp90 inhibitors.
Patent application WO2006/095783 (Nippon Kayaku) claims a family of triazoles as Hsp90 inhibitors.
Patent application WO2006101052 (Nippon Kayaku) claims a family of acetylenic derivatives as Hsp90 inhibitors.
Patent application WO2006105372 (Conforma Therapeutics) claims a family of alkynyl pyrrolo[2,3-d]pyrimidines as Hsp90 inhibitors.
Patent application WO2006101052 (Nippon Kayaku) claims a family of acetylenic derivatives as Hsp90 inhibitors.
Patent application WO2006105372 (Conforma Therapeutics) claims a family of alkynyl pyrrolo[2,3-d]pyrimidines as Hsp90 inhibitors.